We have recently discovered that rat, mouse, and human DATs are modified by palmitoylation, the covalent attachment of palmitic acid. This is the first demonstration of lipid modification for DAT or related neurotransmitter transporters, and significantly impacts our view of transport regulatory mechanisms. Palmitoylation is reversible and dynamic, conferring the ability of proteins to respond to physiologic signals and participate in regulatory processes in a manner analogous to phosphorylation. Our preliminary studies indicate that palmitoylation strongly regulates DA transport capacity and opposes DAT degradation. These novel findings suggest that palmitoylation has high potential to impact dopaminergic signaling in both short- and long-term manners that may be relevant to dopaminergic dysregulation in drug abuse and neurologic disease states. Preliminary studies have identified one of two (or more) palmitoylation sites in rat DAT, with early results consistent with different functions attributable to modification of the different sites. Many major questions related to DAT palmitoylation remain to be addressed. At the most basic level these include identification of the enzymes catalyzing palmitoylation/depalmitoylation, identification of the sites modified, and verification of functions associated with each of these conditions, for both rat and human transporters. Our long-term goal is to understand the regulatory processes involved in modulating DAT activity and to establish causative links in dysfunction of these processes to human neurological disorders and drug abuse. The objective of this proposal is to perform fundamental analyses of the role of palmitoylation in modulation of rat and human DAT function that will lay the groundwork for future studies relevant to understanding DAT activity in normal and pathophysiologic conditions. Our central working hypothesis is that palmitoylation of DAT increases DA transport kinetics and decreases DAT degradation, thereby functioning as a mechanism that would lead to increased DAT transport capacity. To test this hypothesis we propose two specific aims: 1) Identify the enzymes that palmitoylate and depalmitoylate rat and human DAT and assess associated transporter function;and 2) Identify palmitoylation sites in rat and human DAT and assess transporter function associated with modification of these specific sites. For the first aim we will employ overexpression and siRNA knockdown of enzymes that catalyze palmitate addition and removal. For the second aim we will utilize site- directed mutagenesis of expressed transporters, in conjunction with a peptide-mapping analysis of rat striatal DAT to identify the residues palmitoylated in the native protein. This significant research will be performed by undergraduate and graduate students and is expected to substantially advance our understanding of the role of this previously unknown lipid modification on DAT function, and may provide insights into dysregulation of DAT activities leading to dopaminergic disease or drug abuse. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because activity of the dopamine transporter (DAT) is essential for normal dopaminergic neurotransmission and defects in its regulation may be involved with psychiatric and neurodegenerative dopamine related disorders such as schizophrenia, attention deficit disorder, Parkinson's disease, and cocaine and methamphetamine addiction. Thus, the proposed study of regulation of dopamine transporter function by post-translational palmitoylation is relevant to NIH's mission that pertains to developing fundamental knowledge that may provide insights into intervention and prevention of dopamine related diseases including drug abuse.